Numerous conditions in the neonate result in cholestasis, or impairment of bile flow. Understanding biliary development is key to understanding the mechanisms by which cholestatic diseases are caused, yet fairly little is known regarding biliary development at a molecular level. We use the zebrafish to study biliary development, as many features of biliary development in mammals are conserved in zebrafish. The goal of this proposal is gain a greater understanding of biliary development using zebrafish mutants. The zebrafish mutant duct-trip, in which methylation is globally inhibited, demonstrates abnormal biliary development. Inhibition of DNA methylation by chemical or genetic means produces similar bile duct defects, suggesting that inhibition of DNA methylation impairs biliary development. Inhibition of DNA methylation would be expected to derepress expression of genes normally quiescent in biliary cells at this stage of development. One goal of this proposal is to determine which genes are upregulated due to inhibition of DNA methylation in duct-trip. More recently, we have started to examine the mutant pekin, which demonstrates abnormal biliary development and skin hypopigmentation, and is associated with ultrastructural changes consistent with abnormal intracellular vesicular transport in bile duct cells. Another goal of this proposal is to genetically characterize pekin. Finally, we will continue our mutagenesis screen for new mutants that demonstrate abnormal biliary function and development. These studies will address genetic and epigenetic aspects of biliary development in zebrafish;this will strengthen the framework of molecular understanding of biliary development. Many of these genes are likely to be involved in mammalian biliary development;abnormalities in these genes may be associated with specific neonatal biliary diseases.